Asymmetry of lipid organization in cholinergic synaptic vesicle membranes

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Abstract
The lipid composition of purified Torpedo ocellata cholinergic synaptic vesicles was determined, and their distribution between the inner and outer leaflets of the vesicular membrane was investigated. The vesicles contain cholesterol and phospholipids at a molar ratio of 0.63. The vesicular phospholipids are as follows (mol% of total phospholipids): phosphatidylcholine (40.9); phosphatidylethanolamine (24.6); plasmenylethanolamine (11.5); sphingomyelin (12); phosphatidylserine (7.3); phosphatidylinositol (3.7). The asymmetry of the synaptic vesicle membranes was investigated by 2 independent approaches: determining accessibility of the amino lipids to the chemical label trinitrobenzenesulfonic acid (TNBS), and determining accessibility of the vesicular glycerophospholipids to phospholipase C (Bacillus cereus). TNBS rendered the vesicles leaky and cannot be used reliably to determine the asymmetry of T. ocellata synaptic vesicle membranes. Incubation of the vesicles with phospholipase C (B. cereus) results in biphasic hydrolysis of the vesicular glycerophospholipids. About 45% of the phospholipids are hydrolyzed in < 1 min, during which no vesicular acetylcholine is released. In the 2nd phase, the hydrolysis of the phospholipids slows down markedly and is accompanied by loss of all the vesicular acetylcholine. The lipids hydrolyzed during the 1st phase were those comprising the outer leaflet. Analysis of the results thus obtained indicates that the vesicular membrane is asymmetric: all the phosphatidylinositol, 77% of the phosphatidylethanolamine, 47% of the plasmenylethanolamine and 58% of the phosphatidylcholine resided in the outer leaflet. Since phosphatidylserine is a poor substrate for phospholipase C (B. cereus), its distribution between the 2 leaflets of the synaptic vesicle membrane is only suggestive.